Myoglobin and hemoglobin have been and will continue to paradigms for developing our general understanding of protein structure and function. The work propose here will provide crystallographic data on the interaction of ligands of different sizes and shapes with myoglobin and hemoglobin, and examine the structural effects of changes in key amino acid side chains involved in ligand binding. key determinates of the affinity of proteins for their ligands include covalent and hydrogen bonds, electrostatic forces, nonbonded steric and van der Waals interactions, solvent, entropic affects. To date, non complete accounting of all of these "forces" has been made for ligand binding to any protein. Because of its small size, relative simplicity, and extensive experimental study, myoglobin is an ideal protein for this endeavor. Protein with the amino acid sequence of sperm whale myoglobin has been produced in E. coli by Dr. Sligar and co-workers. The only significant difference is that the initiator methionine remains after translation. This expression system allows the rapid generation of mutant proteins to be studied in the proposed research. The structure of the "native" protein has recently been determined so that the successful bacterial synthesis of myoglobin has been verified. Two amino acid side chains are critical for ligand entry and binding in myoglobin. These amino acids are in the distal pocket at positions 64 (HisE7) and 68 (ValE11) in the sequence. The proposed high resolution X- ray crystallographic structure determinations of the deoxy forms of the HisE7->Gly and HisE7->Phe mutants will provide key data on the steric factors that affect ligand entry to the binding pocket of myoglobin. The role of these side chains in the determination of ligand specificity will also b studied by determination of the structures of carbonmonoxy and oxy forms of HisE7->Gly, HisE7->Gln, HisE7->Leu and HisE->Phe mutants, and the carbonmonoxy structures of ValE11->Ala, ValE11->Ile and ValE11->Phe. The effect of different sizes and shapes of ligands on the native forms of myoglobin and hemoglobin will also be examined by determining the structures of several alkyl isocyanide complexes of these proteins. The resulting "strain" on the proteins will indicate which parts of the binding pocket are involved in discrimination between different ligands. The end result of the proposed research will be a better understanding of the details of ligand binding in heme proteins. Molecular dynamics calculations are designed to simulate the detailed dynamic behavior of proteins, and to quantitatively predict some of the forces enumerated above. For these calculations to be meaningful, however, they must be carefully checked against experimental data whenever possible. As the structures of the various myoglobin-ligand complexes are determined, we will use molecular dynamics programs to attempt a rationalization of the changes in structure with the changes in kinetic and thermodynamic measurements.